Sphingolipids are essential in all eukaryotic cells and alterations in sphingolipid metabolism occur in many disease processes. Although a variety of cellular functions for sphingolipids and their metabolites have been proposed, little is known about their biosynthesis and physiological roles. Sphingolipid synthesis in the model eukaryote, S. cerevisiae will be investigated using a combined genetic/biochemical approach. A collection of mutants (csg2, scs1-scs7) with defects in sphingolipid metabolism has been isolated, and will be used to isolate the wild type genes required for sphingolipid synthesis. The scs mutants will be transformed with a genomic library and the wild type genes cloned by complementation of the scs phenotype. The function of each SCS gene will be probed by determining the biochemical consequence of disrupting the gene, by comparing the amino acid sequence of the SCS gene products with the protein data base, by investigating the epistatic relationships between scs, csg, sec and pmr mutants, and by identifying the cellular location of the gene products. Determination of the epistatic relationships between the csg2, scs, sec, and pmr mutants will provide insight into the biosynthetic pathway (both the order of action of the biosynthetic enzymes and their cellular location). Suppressor analysis of scs genes will be used to identify other genes and proteins in sphingolipid synthesis not found in the CSG and SCS collections. Since our scs mutant collection consists of several alleles of each scs gene, mutations that alter the activity in interacting proteins as well as those that bypass the scs gene function altogether will be identified. The results of these experiments should provide valuable information about the genes and proteins that catalyze and regulate sphingolipid synthesis as well as the roles of sphingolipids in all eukaryotic cells.